Available online at www.sciencedirect.
com
Unwinding RNA’s secrets: advances in the biology, physics, and
modeling of complex RNAs
Vincent B Chu2 and Daniel Herschlag1
The rapid development of our understanding of the diverse
biological roles fulfilled by non-coding RNA has motivated
interest in the basic macromolecular behavior, structure, and
function of RNA. We focus on two areas in the behavior of
complex RNAs. First, we present advances in the
understanding of how RNA folding is accomplished in vivo by
presenting a mechanism for the action of DEAD-box proteins.
Members of this family are intimately associated with almost all
cellular processes involving RNA, mediating RNA structural
rearrangements and chaperoning their folding. Next, we focus
on advances in understanding, and characterizing the basic
biophysical forces that govern the folding of complex RNAs.
Ultimately we expect that a confluence and synergy between
these approaches will lead to profound understanding of RNA
and its biology.
Addresses
1
Department of Biochemistry, Stanford University, B400, Beckman
Center, Stanford, CA 94305, United States
2
Department of Applied Physics, Stanford University, GLAM,
McCullough 318, 476 Lomita Mall, Stanford, CA 94305,
United States
Corresponding author: Chu, Vincent B (vincentc@stanford.edu) and
Herschlag, Daniel (herschla@stanford.edu)
Current Opinion in Structural Biology 2008, 18:305–314
This review comes from a themed issue on
Nucleic acids
Edited by Jennifer Doudna and Joseph Puglisi
0959-440X/$ – see front matter
# 2008 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.sbi.2008.05.002
The explosion in our knowledge about non-coding RNA
(ncRNA) – RNA that is not translated into protein – has
expanded interest in RNA beyond the traditional RNA
community. Diverse ncRNAs include the recently dis-
covered riboswitches, whose novel gene-regulation func-
tion is induced by the binding of small metabolites [1];
most of the so-called ‘Human Accelerated Regions’
(HAR) of the human genome, whose recent evolution
may be linked to the emergence of the human brain [2];
and many ncRNAs of unknown function [3]. These
discoveries underscore the need to understand the funda-
mental macromolecular behavior of RNA, its structure
and dynamics, and how these RNAs are handled and
exploited in biology.
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Study of catalytic RNAs, which allows detection of correct
folding via activity measurements, has established basic
thermodynamic and kinetic properties of RNA folding.
Large catalytic RNAs such as the group I intron from
Tetrahymena thermophila and the RNase P RNA from
Bacillus subtilis fold at vastly different rates under differ-
ent conditions upon addition of Mg2+ and have been
shown to fold via multiple parallel pathways, in which
different molecules follow different routes with different
rates and intermediates, to a common final folded struc-
ture [4,5]. These observations support the common view
that RNAs traverse rugged energy landscapes as they fold
[6,7]. However, little is known about the true shape of
these landscapes, and our understanding of the under-
lying physical behavior of RNA is severely limited.
The rugged folding landscape predisposes RNA to form-
ing long-lived misfolded states. Indeed, essentially all
functional RNAs studied in vitro have been found to form
such alternative states, from alternative tRNA folds dis-
covered in the 1960s [8–10] to a recently suggested
topological isomer of a group I intron that takes hours
or days to refold [7,11].
RNA’s propensity to misfold has been attributed to its
intrinsic ‘character flaws’: the low information content of
its four side chains (relative to the 20 for proteins), the
sequestration of these side chains in helices (relative to
the outward-facing side chains in peptide beta sheets and
alpha helices), the promiscuous ability of any base to form
multiple hydrogen bonds with any other base, and the
high stability of stacking interactions of rigidly oriented
groups upon the cyclic base (relative to the more flexible
protein side chains) [12,13].
It has been suggested that the problems encountered by
RNA provided an inroad for peptides and proteins in the
presumed early RNA world [13–15]. RNA chaperones,
proteins that can reorient RNA structures, presumably
evolved to address this misfolding, and considerable
evidence for the existence and roles of RNA chaperones
has accumulated over the past decade [16–19].
In this review, we first focus on recent advances in the
field of chaperone-assisted RNA folding—one of the
more complex processes in RNA biology. We then turn
to recent conceptual and experimental advances in un-
derstanding the basic physical forces that underlie com-
plex RNAs and RNA/protein complexes. We end with a
brief discussion of recent technical advances that will be
crucial for developing a deep energetic, dynamic, and
Current Opinion in Structural Biology 2008, 18:305–314
306 Nucleic acids
structural understanding of RNA and, ultimately, for
drawing profound connections between these physical
properties and behaviors and the biology of RNAs and
RNA/protein complexes.
By necessity, more has been omitted than included in this
short review. Fortunately, there are insightful reviews in
these omitted areas (e.g. [20–28]). It is a remarkable
testament to those in the rather small world of RNA
folding and function that so much progress has been
made in the relatively short time of modern studies of
RNA folding and dynamics.
How ATP-dependent RNA chaperones assist
folding
The predisposition of RNA for misfolding or slow folding
under physiological conditions poses serious challenges
for RNA in vivo [7,13]. Paradoxically, some RNA struc-
tural elements seem to exacerbate misfolding; studies of
the group I intron from Tetrahymena have shown that its
P5abc domain stabilizes misfolded intermediates and
dramatically slows refolding of these intermediates into
the native state [11,29,30]. P5abc’s detrimental effect on
folding kinetics is balanced by its ability to confer thermo-
dynamic stability to the native state and enhance its
catalytic activity [31,32]. RNA chaperones that rescue
misfolded structures allow an escape from these opposing
demands for catalytic and folding efficiency by relieving
selective pressure against slow folding or misfolding
RNAs.
Members of the DExD/H-box protein family, principally
from the major subfamily DEAD-box (the eponymous
DEAD motif is one-letter code for the amino acid
sequence Asp-Glu-Ala-Asp), are involved in virtually
every cellular process involving RNA, ranging from spli-
cing, transport, localization, translation, and decay [23,33].
Although DEAD-box proteins have been casually
referred to as ‘RNA helicases’ on the basis of their
structural similarity to DNA helicases, they are unlikely
to processively unwind helices, as RNA rarely contains
the long helical stretches found in genomic DNA.
Furthermore, functional RNAs often require confor-
mational transitions other than simple helical rearrange-
ments. Since DEAD-box proteins may function
differently from canonical DNA helicases, they have
been less judgmentally referred to as RNA-dependent
ATPases or RNA ‘unwindases’ [34,35,36].
The discovery of DEAD-box proteins that promote spli-
cing in the well-characterized group I and II introns
opened the door for recent mechanistic breakthroughs
[18,37,38,39]. Interestingly, these DEAD-box proteins
were shown to be interchangeable to a substantial extent,
demonstrating that they function rather promiscuously.
Deletion of Mss116p, a mitochondrial DEAD-box protein
from Saccharomyces cerevisiae, adversely affected the spli-
Current Opinion in Structural Biology 2008, 18:305–314
cing of group I and II introns in vivo; these deleterious
effects could be largely rescued by the expression of a
related DEAD-box protein, CYT-19 from Neurospora
crassa [38]. Further, Mss116p and CYT-19 were shown
to promote group I and group II intron splicing in vitro, as
was the cytosolic DEAD-box protein, Ded1p [37,38,39].
Each of these proteins was also shown to unwind RNA
helices non-specifically in an ATP-dependent manner
[36,37,39–41].
The ability of these DEAD-box proteins to promote
proper folding of structurally diverse RNAs and their
common structural features suggest that mechanistic
details obtained for one particular DEAD-box protein
and RNA substrate may yield general insights for a wide
variety of DEAD-box proteins. Recently, the mechanism
of CYT-19 has been probed in the folding and function of
a ribozyme derived from the Tetrahymena group I intron.
CYT-19 was shown to act non-specifically, disrupting
both misfolded and native forms of the ribozyme. How-
ever, the efficiency of unfolding activity was dependent
on the stability of the RNA species, with CYT-19 unfold-
ing the misfolded RNA at least 50-fold faster than the
native ribozyme [42,43].
Unexpectedly, CYT-19 was also able to unwind the base
paired P1 duplex, formed between the ribozyme and its
oligonucleotide substrate, much more efficiently than the
same duplex free in solution, demonstrating that local
unwinding activity was enhanced by the presence of the
larger intron structure. Further, unwinding activity was
abolished if tertiary contacts were formed between P1
and the intron. This curious result immediately suggested
a physical model for the chaperone activity of CYT-19
and other DEAD-box proteins. Chaperones function by
first binding non-specifically to structured RNA, then
rearranging RNA structure by unwinding double-
stranded regions and perhaps other accessible structures
[36]. This general model was also supported by a clever
experiment in which the unwinding activity of Ded1p
was shown to be enhanced by an ssRNA tethered through
a streptavidin linker to a model RNA duplex [44].
These and related results lead to a general model for RNA
chaperone function (Figure 1) [40,45]. The proteins do
not recognize any specific structural features of their
target RNAs; unfolding efficiency is dependent on the
accessibility of the target. If a structural element is loosely
associated with the rest of the RNA, it is easy to ‘grab’ and
unwind. This model elegantly explains the enhanced
activity of CYT-19 on misfolded relative to native
RNA, as properly folded structure is more compact and
will have fewer conformational excursions of its structural
elements.
A consequence of non-specific unfolding activity is that
DExD/H-box proteins can drive folding away from
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 Advances in RNA structure and function Herschlag and Chu 307

Figure 1

Model for general ATP-driven chaperone activity by CYT-19 and other DEAD-box proteins. CYT-19 first binds to structured RNAs using a binding site
(RBD). Binding of CYT-19 then allows for structural rearrangement through local, non-processive unwinding of helical elements such as the P1 duplex
(green and red strands). In general, the accessibility of the targeted structural element determines CYT-19’s unwinding efficiency. Reprinted from
[36]. Copyright 1993–2008 by The National Academy of Sciences of the United States of America, all rights reserved.

thermodynamic equilibrium, and such effects have
recently been demonstrated for Ded1p and CYT-19
[42,43,46]. These results suggest that, subsequent to
each ATP-driven unfolding event, the RNA repartitions
between folding forms based on the kinetics of this
partitioning rather than the stability of the ultimate
folded states. If the stability and accessibility differences
between the alternative folded states of the RNA are not
too large, the chaperone can unfold both species and allow
accumulation of a less stable but kinetically preferred
state.
The ability of DExD/H-box proteins to perturb RNAs
away from thermodynamic equilibrium may also have
profound biological implications beyond its implications
for the chaperone mechanism. Structured RNAs are
involved in complex, multi-step processes, such as pre-
mRNA splicing and translation. Although the details of
these processes remain elusive, it is plausible that DExD/
H-box proteins capture the energy of ATP hydrolysis to
drive the transient formation of thermodynamically
unstable states that are part of the normal reaction cycle.
Although non-specific DEAD-box chaperones could be
important for these processes, presumably most of the
required structural changes are mediated by dedicated
DExD/H-box proteins, which use specific RNA or
protein interactions to position themselves within com-
plexes for rearrangement of the desired structural
element [47–49].
A future challenge in this area is dissecting the individual
reaction steps in complex RNA-mediated processes and
unraveling the physical roles played by DExD/H-box
proteins. The recently attained ability to follow pre-
mRNA splicing by FRET at the single molecule level
represents a technical breakthrough toward this import-
ant goal [50].
Simple forces underlying the complex
behavior of RNA
As noted in the Introduction, one of the primary lessons
from prior work is that RNA folding is highly complex.
However, work from several groups, aimed at understand-
ing the underlying physical properties of RNA and the
behavior of RNA components, suggests the feasibility and
potential of a ‘bottom up’ approach to RNA folding.
Figure 2 presents a simple hierarchy of forces and features
that underlie the folding of all RNAs. Understanding the
underlying behavior of each component, from theory or
empirical observation, would provide the foundation for
describing and understanding of the behavior of complex
RNAs. In this section, we explain this hierarchy and
emphasize some recent foundational advances in our
understanding.
The formation of secondary structure, encoded in RNA
sequence, is typically an early step in folding. The
stability of RNA secondary structure and the fact that
formation of secondary and tertiary structure is often
temporally separated simplifies the consideration of the
folding process [51] (see also [28]). However, RNA sec-
ondary structure topology is more complex than the
topology of proteins, and understanding this complexity

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308 Nucleic acids
Figure 2
A schematic of the hierarchy of forces that determine RNA folding.
Electrostatic repulsion between helices (depicted as cylinders) dictates
the energetic cost of forming a compact structure. Electrostatic
screening from an associated ion atmosphere (blue dots) mitigates this
cost (a). Junctions that join helical regions bias the relative positions of
the helices that they join, bringing different regions of secondary
structure in close proximity (b). Recurring tertiary motifs bind secondary
structure elements together and stabilize the final folded state. Some
motifs, such as the tetraloop/tetraloop receptor (whose secondary
structure is pictured) are highly conserved and ubiquitous throughout
RNA structure (c).
and the folding constraints introduced by secondary
structure topology will require advanced modeling and
experimental approaches [52,53].
In the absence of other forces, the predominant force on
secondary structure helices (depicted in Figure 2 as
cylinders) is the enormous inter-helical electrostatic
repulsion due to the polyelectrolyte nature of the
RNA. Draper has written extensively on the importance
of the ‘ion atmosphere’ that surrounds any polyelectrolyte
and has noted that the ions in this atmosphere are likely to
provide the dominant electrostatic contribution to the
folding energetics, far greater than any ions bound to
specific sites within the RNA [54,55].
Nevertheless, the ion atmosphere has been a difficult
concept for experimentalists to grasp, both because of
limited familiarity with the underlying theory and
because of our inability to directly visualize this dynamic
sheath of ions via X-ray crystallography or other direct
approaches. Thus, the recent ability to decipher the shape
of this atmosphere by anomalous small angle X-ray scat-
tering provided an unusually direct experimental window
into properties of the atmosphere [56,57]. Further, the
contents of the atmosphere have recently been quanti-
tatively dissected, by essentially counting the ions associ-
Current Opinion in Structural Biology 2008, 18:305–314
ated with a nucleic acid under a variety of ionic
conditions. This technique allows RNA systems to be
characterized stoichiometrically in terms of the number of
each cation and anion species present in the ion atmos-
phere [58].
The next challenge is the calculation of electrostatic
energies, as these energies determine the energetic pen-
alty of assembling helices in densely packed arrange-
ments. Most simply, helices at low ionic concentrations
repel; at higher ionic concentration, this repulsion is
reduced owing to the enhanced screening, allowing
helices to approach one another (Figure 2a). However,
some have proposed the existence of attractive forces in
polyelectrolyte systems under certain ionic conditions
that may be responsible for the coalescence of helices
in the folded state [59,60].
While simulation experiments and the aggregation of
DNA and RNA in ethanol during standard precipitation
procedures and other conditions demonstrate the exist-
ence of such attractive forces under some conditions, their
exact magnitude and significance for folding in vitro was
unknown [61,62]. The magnitude of these attractive
forces was tested with a simple model: two DNA helices
tethered together by a neutral polyethylene glycol tether.
Small angle X-ray scattering (SAXS) revealed no signifi-
cant attractive forces under standard in vitro folding
conditions [63]. Thus, the observed ‘electrostatic col-
lapse’ of large RNAs upon addition of ions appears to
arise from a screening of repulsive force rather than the
action of an attractive one (e.g. [64–66]).
There have also been recent advances in understanding
and quantitating electrostatic forces. The predominant
electrostatic theory used by biochemists has been Pois-
son-Boltzmann (PB) theory, a computationally con-
venient method for calculation of approximate
electrostatic forces [67]. PB theory neglects potentially
important excluded volume effects from ion-size and
positional correlation effects from ion–ion electrostatic
interactions. In particular, correlation effects are pre-
dicted to be more important for multivalent ions.
Simulation has suggested that PB theory is inadequate for
describing electrostatic energies for nucleic acids in the
presence of divalent ions such as Mg2+, a conclusion
supported by experiments assessing the effects of ions
on DNA duplex stability ([68,69] and references therein).
However, the opposite conclusion has been drawn from
fitting RNA folding transitions to PB predictions [70–74].
Ion-counting and SAXS experiments have provided a
simple test for these effects and have revealed effects
of ion size and valence that are not accounted for by PB
theory, with deviations up to an order of magnitude in the
systems investigated [58,75]. Advances in theory and
simulation are required to account for these discrepancies
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and obtain accurate electrostatic energies for use in dis-
secting RNA folding and RNA/protein energetics [76,77].
The junctions in secondary structure bias the confor-
mations of the helices they join (Figure 2b). Ground-
breaking work by Lilley on Holliday junctions – four-way
DNA junctions involved in recombination – and junctions
found in structured RNAs has shown that these junctions
position their attached helices into preferred confor-
mations [78–80]. Such preferred geometries are integral
to the adoption of stable, active structure by functional
RNAs. Ha and Lilley have used single molecule FRET to
probe the underlying kinetics and thermodynamics of
hairpin ribozyme folding, showing that the conformation-
al dynamics of its junction accelerate folding and promote
formation of its active site [81]. An important challenge
for understanding and predicting RNA structure and
dynamics remains to interrogate the behaviors and struc-
tures of isolated RNA junctions to reveal their intrinsic
behaviors and then how these behaviors determine and
are modulated by the rest of the RNA molecule in which
they are found. The development of a database of RNA
junctions extracted from structured RNAs will undoubt-
edly help advance this goal [82].
RNAs often utilize motifs for ‘gluing’ RNA secondary
structure together to fold to precise tertiary structures
(Figure 2c). Long-range interactions in RNA are often
found by phylogenetic covariation, beginning with tRNA
whose tertiary structure was first modeled in 1969 [83].
The tetraloop/tetraloop receptor motif is perhaps the
most famous RNA motif. It was first identified by phy-
logeny in group I and II introns by Michel, Westhof, and
colleagues and observed for the first time in the seminal
crystal structure of the P4–P6 domain of the Tetrahymena
group I intron, in which this motif reinforces the side-by-
side packing architecture of RNA helices [84–86]. This
motif and others are ubiquitous in the ribosome and other
structured RNAs [87–91]. We wish to emphasize that
information on motif partners and secondary structure –
determined from phylogeny – provides a powerful tool in
modeling RNA structure. Westhof has developed and
exploited this tool to create impressive first generation
three-dimensional models for many structured RNAs
[92].
Early studies emphasized special roles for Mg2+ in RNA
folding and function. However, some RNAs have been
shown to fold without Mg2+ [93–95]. Some, such as the
hairpin and hammerhead ribozymes, can even function
in its absence [96,97]. A recent dissection of folding the
P4–P6 domain from the Tetrahymena group I intron has
helped clarify the roles of metal ions in RNA folding
and, more generally, the underlying energetics of fold-
ing [94]. This RNA can adopt a compact shape with its
tetraloop motif formed in the presence of monovalent
ions alone.
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Advances in RNA structure and function Herschlag and Chu 309
Instead of a special role for Mg2+, folding of P4–P6 and of
RNAs in general can be accounted for by a simple balance
of forces and interactions outlined in Figure 2. Unfavor-
able electrostatic repulsion and loss of conformational
entropy in the folded state resist folding, whereas junc-
tion preferences and tertiary motif formation favor it.
Electrostatic forces are modulated by ion concentration
and valence; thus, even monovalent cations can mitigate
electrostatic repulsion to allow folding to be favorable if
present at sufficient concentrations. Fewer divalent ions
are needed since they are much more effective at charge
screening (localizing a single divalent ion around RNA is
entropically less costly than two monovalents). Specific
divalent ion binding sites – if present in the folded RNA –
contribute additional stability. The contributions from
specifically bound divalent cations have been isolated by
saturating the ion atmosphere with monovalent cations
and following the binding of two specific divalent metal
ions to the core of the P4–P6 RNA [98,99] (see also [100]).
RNA: present and future
We have highlighted some of the conceptual and exper-
imental advances over the past few years and some of the
future challenges. In this section, we briefly note some
additional methodological advances in the context of
challenges that remain in unraveling the structure, ener-
getics, and dynamics of RNA.
Single molecule force experiments have far exceeded
simple proof-of-principle experiments and have been
used to elucidate the folding thermodynamics and
kinetics for several RNAs [101–104,105,106]. This
approach has answered one of the most fundamental
questions in nucleic acid structure formation, providing
direct evidence for the long-held nucleation model for
duplex involving formation of two to three base pairs
before rapid formation of the remainder of the duplex
[107]. Further, it has been shown that force unfolding data
can be deconstructed into a complete energy landscape
[108]. It will be fascinating to see if this approach can be
applied to increasingly complex RNAs.
There have been important advances in studying the
kinetics of the folding of medium and large RNAs.
Time-resolved hydroxyl radical footprinting experiments
at millisecond resolution are now routinely conducted
using standard rapid-quench devices, without the need
for synchrotron radiation to generate radicals [109].
Thus, a large body of kinetic data on RNA folding at
the residue level is readily obtainable. This large body of
data can be analyzed in minutes instead of hours, using
freely available semi-automated software (https://simtk.
org/home/safa) [110]. Still higher throughput data acqui-
sition is now possible using capillary electrophoresis and a
software analysis package called CAFA [111]. Parallel
advances have been made with a new and powerful
footprinting approach called ‘SHAPE’, which shows
Current Opinion in Structural Biology 2008, 18:305–314
310 Nucleic acids
protection data for residues in secondary and tertiary
structures [112–114]. New kinetic modeling approaches
have greatly simplified and systematized the develop-
ment of kinetic models from large sets of footprinting
data; such an approach has been recently applied to
characterize the folding pathways of wild type and mutant
Tetrahymena group I intron [115,116].
Early single molecule FRET studies on RNA folding
kinetics revealed a wealth of information inaccessible to
traditional pre-steady state kinetic methods, demonstrat-
ing the utility of these studies [117–119]. Since then,
there has been an explosion of single molecule fluor-
escence studies on RNA [120]. One of the most puzzling
observations in these studies is of molecular ‘memory’—
the fact that different molecules tend to display similar
kinetic behavior over long time scales that differ from that
of other molecules of the same sequence [81,121].
Future work will undoubtedly address the basis for such
quixotic behavior by single molecules.
RNA structures remain difficult to obtain relative to
protein structures despite the solution of the ribosome
structure other RNA structures. Piccirilli and co-workers,
following on observations that proteins may help RNA
crystallization, have pioneered a potentially high-
throughput approach for crystallization of RNA/antibody
complexes [122]. Nevertheless, many important RNA
structures may be partially or intrinsically unstructured
and thus resistant to crystallization, necessitating the
development of new approaches to RNA structure. Med-
ium resolution structural information is now obtainable
from a method that allows pairwise distance constraints to
be read from a single polyacrylamide gel. This method
has been used to obtain structural information about a
molten globule-like state of the P4–P6 RNA [123]. Also,
additional modeling approaches are needed for RNA, and
a first pass fragment assembly method, following the
highly successful approach of David Baker for protein
structure prediction, has been pioneered for RNA [124].
The intrinsically dynamic behavior of RNA that compli-
cates structure determination has also been highly resist-
ant to study. However, recent advances in NMR
techniques over the past two years are now providing
an unprecedented view into the structural dynamics of
RNA [125–127]. Particularly powerful is the ability to
determine the amplitude and relative directionality of the
internal motion of helical domains through residual dipo-
lar coupling measurements [128,129].
In this review we have emphasized advances in mechan-
istic and physical understanding using model RNAs of
varying complexity. In parallel, the biology community is
increasingly recognizing the diverse roles of RNA in basic
gene expression and regulation. We suspect that bio-
logical and physical approaches to understanding RNA
Current Opinion in Structural Biology 2008, 18:305–314
will converge in the coming decade as biological systems
are reconstituted and characterized and the tools for un-
derstanding RNA are developed and perfected. While
there is much work ahead, we eagerly anticipate that union.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
The authors thank and apologize to the many investigators in the RNA
world whose work has contributed greatly to our overall understanding of
RNA but could not be cited owing to space limitations. It is truly a
testament to this vigorous community that so much has been accomplished
of note in the recent years. We would like to thank Yu Bai, Max Greenfeld,
Jan Lipfert, Jungwon Park, Adelene Sim, and Sergey Solomatin for critical
discussion and insightful comments and all of our collaborators. Our work on
RNA folding is supported by NIH grant PO1 GM066275. VBC is supported
by a Pfizer Bio-X Graduate fellowship. We have adopted an agnostic view in
highlighting references to our own work to avoid the moral dilemma of
assigning ‘significance stars’ to our own work.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1. Mandal M, Breaker RR: Gene regulation by riboswitches. Nat
Rev Mol Cell Biol 2004, 5:451-463.
2. Pollard KS, Salama SR, Lambert N, Lambot MA, Coppens S,
Pedersen JS, Katzman S, King B, Onodera C, Siepel A et al.: An
RNA gene expressed during cortical development evolved
rapidly in humans. Nature 2006, 443:167-172.
3. Eddy SR: Non-coding RNA genes and the modern RNA world.
Nat Rev Genet 2001, 2:919-929.
4. Pan T, Fang X, Sosnick T: Pathway modulation, circular
permutation and rapid RNA folding under kinetic control. J Mol
Biol 1999, 286:721-731.
5. Russell R, Zhuang X, Babcock HP, Millett IS, Doniach S, Chu S,
Herschlag D: Exploring the folding landscape of a structured
RNA. Proc Natl Acad Sci U S A 2002, 99:155-160.
6. Thirumalai D, Lee N, Woodson SA, Klimov D: Early events in RNA
folding. Annu Rev Phys Chem 2001, 52:751-762.
7. Treiber DK, Williamson JR: Exposing the kinetic traps in RNA
folding. Curr Opin Struct Biol 1999, 9:339-345.
8. Gartland WJ, Sueoka N: Two interconvertible forms of
tryptophanyl sRNA in E. coli. Proc Natl Acad Sci U S A 1966,
55:948-956.
9. Adams A, Lindahl T, Fresco JR: Conformational differences
between the biologically active and inactive forms of a transfer
ribonucleic acid. Proc Natl Acad Sci U S A 1967, 57:1684-1691.
10. Ishida T, Sueoka N: Effect of ambient conditions on
conformations of tryptophan transfer ribonucleic acid of
Escherichia coli. J Biol Chem 1968, 243:5329-5336.
11. Russell R, Das R, Suh H, Travers KJ, Laederach A, Engelhardt MA,
Herschlag D: The paradoxical behavior of a highly structured
misfolded intermediate in RNA folding. J Mol Biol 2006,
363:531-544.
12. Sigler PB: An analysis of the structure of tRNA. Annu Rev
Biophys Bioeng 1975, 4:477-527.
13. Herschlag D: RNA chaperones and the RNA folding problem.
J Biol Chem 1995, 270:20871-20874.
14. Karpel RL, Swistel DG, Miller NS, Geroch ME, Lu C, Fresco JR:
Acceleration of RNA renaturation by nucleic acid unwinding
proteins. Brookhaven Symp Biol 1975:165-174.
www.sciencedirect.com

15. Karpel RL, Miller NS, Fresco JR: Mechanistic studies of
ribonucleic acid renaturation by a helix-destabilizing protein.
Biochemistry 1982, 21:2102-2108.
16. Rein A, Henderson LE, Levin JG: Nucleic-acid-chaperone
activity of retroviral nucleocapsid proteins: significance for
viral replication. Trends Biochem Sci 1998, 23:297-301.
17. Lorsch JR: RNA chaperones exist and DEAD box proteins get a
life. Cell 2002, 109:797-800.
18. Mohr S, Stryker JM, Lambowitz AM: A DEAD-box protein
functions as an ATP-dependent RNA chaperone in group I
intron splicing. Cell 2002, 109:769-779.
19. Schroeder R, Barta A, Semrad K: Strategies for RNA folding and
assembly. Nat Rev Mol Cell Biol 2004, 5:908-919.
20. Collins K: The biogenesis and regulation of telomerase
holoenzymes. Nat Rev Mol Cell Biol 2006, 7:484-494.
21. Williamson JR: Assembly of the 30S ribosomal subunit. Q Rev
Biophys 2005, 38:397-403.
22. Lipfert J, Doniach S: Small-angle X-ray scattering from RNA,
proteins, and protein complexes. Annu Rev Biophys Biomol
Struct 2007, 36:307-327.
23. Jankowsky E, Fairman ME: RNA helicases—one fold for many
functions. Curr Opin Struct Biol 2007, 17:316-324.
24. Hammann C, Westhof E: Searching genomes for ribozymes and
riboswitches. Genome Biol 2007, 8:210.
25. Hoogstraten CG, Sumita M: Structure–function relationships in
RNA and RNP enzymes: recent advances. Biopolymers 2007,
87:317-328.
26. Cristofari G, Darlix JL: The ubiquitous nature of RNA chaperone
proteins. Prog Nucleic Acid Res Mol Biol 2002, 72:223-268.
27. Wolin SL, Cedervall T: The La protein. Annu Rev Biochem 2002,
71:375-403.
28. Pan T, Sosnick T: RNA folding during transcription. Annu Rev
Biophys Biomol Struct 2006, 35:161-175.
29. Russell R, Tijerina P, Chadee AB, Bhaskaran H: Deletion of the
P5abc peripheral element accelerates early and late folding
steps of the Tetrahymena group I ribozyme. Biochemistry 2007,
46:4951-4961.
30. Treiber DK, Rook MS, Zarrinkar PP, Williamson JR: Kinetic
intermediates trapped by native interactions in RNA folding.
Science 1998, 279:1943-1946.
31. Doherty EA, Herschlag D, Doudna JA: Assembly of an
exceptionally stable RNA tertiary interface in a group I
ribozyme. Biochemistry 1999, 38:2982-2990.
32. Engelhardt MA, Doherty EA, Knitt DS, Doudna JA, Herschlag D:
The P5abc peripheral element facilitates preorganization of
the Tetrahymena group I ribozyme for catalysis. Biochemistry
2000, 39:2639-2651.
33. Tanner NK, Linder P: DExD/H box RNA helicases: from generic
motors to specific dissociation functions. Mol Cell 2001,
8:251-262.
34. Staley JP, Guthrie C: Mechanical devices of the spliceosome:
motors, clocks, springs, and things. Cell 1998, 92:315-326.
35. Mayas RM, Staley JP: DEAD on. Nat Struct Mol Biol 2006,
13:954-955.
36. Tijerina P, Bhaskaran H, Russell R: Nonspecific binding to
 structured RNA and preferential unwinding of an exposed
helix by the CYT-19 protein, a DEAD-box RNA chaperone. Proc
Natl Acad Sci U S A 2006, 103:16698-16703.
The authors elucidate the mechanism for CYT-19, a DEAD-box RNA
chaperone, finding that its RNA duplex unwinding action is driven by ATP
and enhanced by the presence of surrounding RNA structure. These
results led to the proposal of a model for CYT-19’s activity: non-specific
binding of the chaperone, followed by structural disruption via local
duplex unwinding.
37. Mohr S, Matsuura M, Perlman PS, Lambowitz AM: A DEAD-box
protein alone promotes group II intron splicing and reverse
www.sciencedirect.com
Advances in RNA structure and function Herschlag and Chu 311
splicing by acting as an RNA chaperone. Proc Natl Acad Sci U S
A 2006, 103:3569-3574.
38. Huang HR, Rowe CE, Mohr S, Jiang Y, Lambowitz AM, Perlman PS:
 The splicing of yeast mitochondrial group I and group II introns
requires a DEAD-box protein with RNA chaperone function.
Proc Natl Acad Sci U S A 2005, 102:163-168.
The authors demonstrate the promiscuity of DEAD-box proteins CYT-19
(from Neurospora crassa) and Mss116p (from Saccharomyces cerevisiae)
by showing that splicing reactions in Mss116p-deletion mutants can be
rescued by expression of CYT-19 in vivo.
39. Halls C, Mohr S, Del Campo M, Yang Q, Jankowsky E,
Lambowitz AM: Involvement of DEAD-box proteins in group I
and group II intron splicing. Biochemical characterization of
Mss116p, ATP hydrolysis-dependent and -independent
mechanisms, and general RNA chaperone activity. J Mol Biol
2007, 365:835-855.
40. Grohman JK, Del Campo M, Bhaskaran H, Tijerina P,
Lambowitz AM, Russell R: Probing the mechanisms of DEAD-
box proteins as general RNA chaperones: the C-terminal
domain of CYT-19 mediates general recognition of RNA.
Biochemistry 2007, 46:3013-3022.
41. Yang Q, Jankowsky E: ATP- and ADP-dependent modulation of
RNA unwinding and strand annealing activities by the DEAD-
box protein DED1. Biochemistry 2005, 44:13591-13601.
42. Bhaskaran H, Russell R: Kinetic redistribution of native and
 misfolded RNAs by a DEAD-box chaperone. Nature 2007,
449:1014-1018.
The authors demonstrate the ability of CYT-19, a DEAD-box protein from
Neurospora crassa, to unwind the native form of the Tetrahymena ribo-
zyme, albeit 50-fold less efficiently than the misfolded form. Furthermore,
the ATP-driven unfolding activity actively redistributes the conformational
ensemble of structured RNAs, populating intermediates that otherwise
would be rare at thermodynamic equilibrium.
43. Jankowsky E: Biochemistry: indifferent chaperones. Nature
2007, 449:999-1000.
44. Yang Q, Jankowsky E: The DEAD-box protein Ded1 unwinds
RNA duplexes by a mode distinct from translocating
helicases. Nat Struct Mol Biol 2006, 13:981-986.
45. Yang Q, Del Campo M, Lambowitz AM, Jankowsky E: DEAD-box
 proteins unwind duplexes by local strand separation. Mol Cell
2007, 28:253-263.
The authors demonstrate that DEAD-box RNA chaperones are qualita-
tively different from canonical DNA helicases by examining the activity
Ded1p and Mss116p. They find that these two DEAD-box proteins
unwind RNA helices non-specifically, acting as local ‘strand separators’.
Their lack of processive or directional translocation along helices makes
their unwinding activity distinct from the mechanism of typical DNA
helicases.
46. Yang Q, Fairman ME, Jankowsky E: DEAD-box-protein-assisted
 RNA structure conversion towards and against
thermodynamic equilibrium values. J Mol Biol 2007,
368:1087-1100.
The authors investigate the activity of Ded1p, a DEAD-box protein, in a
model system and observe its ability to populate less stable structures by
converting more stable structures to misfolded ones using the energy
from ATP hydrolysis.
47. Karginov FV, Caruthers JM, Hu Y, McKay DB, Uhlenbeck OC: YxiN
is a modular protein combining a DEx(D/H) core and a specific
RNA-binding domain. J Biol Chem 2005, 280:35499-35505.
48. Silverman E, Edwalds-Gilbert G, Lin RJ: DExD/H-box proteins
and their partners: helping RNA helicases unwind. Gene 2003,
312:1-16.
49. Wang S, Hu Y, Overgaard MT, Karginov FV, Uhlenbeck OC,
McKay DB: The domain of the Bacillus subtilis DEAD-box
helicase YxiN that is responsible for specific binding
of 23S rRNA has an RNA recognition motif fold. RNA 2006,
12:959-967.
50. Crawford DJ, Hoskins AA, Friedman LJ, Gelles J, Moore MJ:
Visualizing the splicing of single pre-mRNA molecules in
whole cell extract. RNA 2008, 14:170-179.
51. Brion P, Westhof E: Hierarchy and dynamics of RNA folding.
Annu Rev Biophys Biomol Struct 1997, 26:113-137.
Current Opinion in Structural Biology 2008, 18:305–314
312 Nucleic acids
52. Cao S, Chen SJ: Predicting RNA folding thermodynamics with a
reduced chain representation model. RNA 2005, 11:1884-1897.
53. Cao S, Chen SJ: Biphasic folding kinetics of RNA pseudoknots
and telomerase RNA activity. J Mol Biol 2007, 367:909-924.
54. Draper DE, Grilley D, Soto AM: Ions and RNA folding. Annu Rev
Biophys Biomol Struct 2005, 34:221-243.
55. Draper DE: A guide to ions and RNA structure. RNA 2004,
10:335-343.
56. Andresen K, Das R, Park HY, Smith H, Kwok LW, Lamb JS,
Kirkland EJ, Herschlag D, Finkelstein KD, Pollack L: Spatial
distribution of competing ions around DNA in solution. Phys
Rev Lett 2004, 93:248103.
57. Das R, Mills TT, Kwok LW, Maskel GS, Millett IS, Doniach S,
Finkelstein KD, Herschlag D, Pollack L: Counterion distribution
around DNA probed by solution X-ray scattering. Phys Rev Lett
2003, 90:188103.
58. Bai Y, Greenfeld M, Travers KJ, Chu VB, Lipfert J, Doniach S,
Herschlag D: Quantitative and comprehensive decomposition
of the ion atmosphere around nucleic acids. J Am Chem Soc
2007, 129:14981-14988.
59. Heilman-Miller SL, Thirumalai D, Woodson SA: Role of counterion
condensation in folding of the Tetrahymena ribozyme. I.
Equilibrium stabilization by cations. J Mol Biol 2001,
306:1157-1166.
60. Murthy VL, Rose GD: Is counterion delocalization responsible
for collapse in RNA folding? Biochemistry 2000, 39:14365-
14370.
61. Grosberg AY, Nguyen TT, Shklovskii BI: Colloquium: the physics
of charge inversion in chemical and biological systems. Rev
Mod Phys 2002, 74:329-345.
62. Bloomfield VA: DNA condensation by multivalent cations.
Biopolymers 1997, 44:269-282.
63. Bai Y, Das R, Millett IS, Herschlag D, Doniach S: Probing
counterion modulated repulsion and attraction between
nucleic acid duplexes in solution. Proc Natl Acad Sci U S A 2005,
102:1035-1040.
64. Buchmueller KL, Webb AE, Richardson DA, Weeks KM: A
collapsed non-native RNA folding state. Nat Struct Biol 2000,
7:362-366.
65. Das R, Kwok LW, Millett IS, Bai Y, Mills TT, Jacob J, Maskel GS,
Seifert S, Mochrie SG, Thiyagarajan P et al.: The fastest global
events in RNA folding: electrostatic relaxation and tertiary
collapse of the Tetrahymena ribozyme. J Mol Biol 2003,
332:311-319.
66. Russell R, Millett IS, Tate MW, Kwok LW, Nakatani B, Gruner SM,
Mochrie SG, Pande V, Doniach S, Herschlag D et al.: Rapid
compaction during RNA folding. Proc Natl Acad Sci U S A 2002,
99:4266-4271.
67. Honig B, Nicholls A: Classical electrostatics in biology and
chemistry. Science 1995, 268:1144-1149.
68. Tan ZJ, Chen SJ: Nucleic acid helix stability: effects of salt
concentration, cation valence and size, and chain length.
Biophys J 2006, 90:1175-1190.
2+
69. Tan ZJ, Chen SJ: RNA helix stability in mixed Na+/Mg
solution. Biophys J 2007, 92:3615-3632.
70. Misra VK, Draper DE: The linkage between magnesium binding
and RNA folding. J Mol Biol 2002, 317:507-521.
71. Misra VK, Shiman R, Draper DE: A thermodynamic framework
for the magnesium-dependent folding of RNA. Biopolymers
2003, 69:118-136.
72. Soto AM, Misra V, Draper DE: Tertiary structure of an RNA
pseudoknot is stabilized by ‘diffuse’ Mg2+ ions. Biochemistry
2007, 46:2973-2983.
73. Grilley D, Soto AM, Draper DE: Mg2+-RNA interaction free
energies and their relationship to the folding of RNA tertiary
structures. Proc Natl Acad Sci U S A 2006, 103:14003-14008.
Current Opinion in Structural Biology 2008, 18:305–314
74. Grilley D, Misra V, Caliskan G, Draper DE: Importance of partially
unfolded conformations for Mg(2+)-induced folding of RNA
tertiary structure: structural models and free energies of Mg2+
interactions. Biochemistry 2007, 46:10266-10278.
75. Bai Y, Chu VB, Lipfert J, Pande VS, Herschlag D, Doniach S:
Experimental and computational reconstruction of the
unfolded state ensemble in nucleic acid folding. 2008,
submitted for publication.
76. Chu VB, Bai Y, Lipfert J, Herschlag D, Doniach S: Evaluation of
ion binding to DNA duplexes using a size-modified Poisson-
Boltzmann theory. Biophys J 2007, 93:3202-3209.
77. Tan ZJ, Chen SJ: Electrostatic correlations and fluctuations for
ion binding to a finite length polyelectrolyte. J Chem Phys 2005,
122:44903.
78. Lilley DM: Structures of helical junctions in nucleic acids. Q Rev
Biophys 2000, 33:109-159.
79. Hohng S, Wilson TJ, Tan E, Clegg RM, Lilley DM, Ha T:
Conformational flexibility of four-way junctions in RNA. J Mol
Biol 2004, 336:69-79.
80. Walter F, Murchie AI, Lilley DM: Folding of the four-way RNA
junction of the hairpin ribozyme. Biochemistry 1998,
37:17629-17636.
81. Tan E, Wilson TJ, Nahas MK, Clegg RM, Lilley DM, Ha T: A four-
 way junction accelerates hairpin ribozyme folding via a
discrete intermediate. Proc Natl Acad Sci U S A 2003,
100:9308-9313.
The authors probe the influence of a naturally occurring four-way junction
in the folding hairpin ribozyme using single molecule spectroscopy
demonstrating that its junction accelerates the folding of the molecule
and the organization of the active site.
82. Bindewald E, Hayes R, Yingling YG, Kasprzak W, Shapiro BA:
 RNAJunction: a database of RNA junctions and kissing loops
for three-dimensional structural analysis and nanodesign.
Nucleic Acids Res 2008, 36:D392-D397.
The authors present a database of RNA junctions extracted from struc-
tural RNA data, offering researchers a centralized collection of data to
perform structural analyses and structure design.
83. Levitt M: Detailed molecular model for transfer ribonucleic
acid. Nature 1969, 224:759-763.
84. Costa M, Michel F: Frequent use of the same tertiary motif by
self-folding RNAs. EMBO J 1995, 14:1276-1285.
85. Michel F, Westhof E: Modelling of the three-dimensional
architecture of group I catalytic introns based on comparative
sequence analysis. J Mol Biol 1990, 216:585-610.
86. Cate JH, Gooding AR, Podell E, Zhou K, Golden BL, Kundrot CE,
Cech TR, Doudna JA: Crystal structure of a group I ribozyme
domain: principles of RNA packing. Science 1996,
273:1678-1685.
87. Noller HF: RNA structure: reading the ribosome. Science 2005,
 309:1508-1514.
A detailed review about the vastly increased amount of structural data
from the ribosome crystal structure and its implications for our under-
standing of RNA folding, structure, and function.
88. Golden BL, Kim H, Chase E: Crystal structure of a phage Twort
group I ribozyme-product complex. Nat Struct Mol Biol 2005,
12:82-89.
89. Torres-Larios A, Swinger KK, Krasilnikov AS, Pan T, Mondragon A:
Crystal structure of the RNA component of bacterial
ribonuclease P. Nature 2005, 437:584-587.
90. Doherty EA, Batey RT, Masquida B, Doudna JA: A universal mode
of helix packing in RNA. Nat Struct Biol 2001, 8:339-343.
91. Nissen P, Ippolito JA, Ban N, Moore PB, Steitz TA: RNA tertiary
interactions in the large ribosomal subunit: the A-minor motif.
Proc Natl Acad Sci U S A 2001, 98:4899-4903.
92. Lehnert V, Jaeger L, Michel F, Westhof E: New loop-loop tertiary
interactions in self-splicing introns of subgroup IC and ID: a
complete 3D model of the Tetrahymena thermophila
ribozyme. Chem Biol 1996, 3:993-1009.
www.sciencedirect.com

93. Rangan P, Woodson SA: Structural requirement for Mg2+
binding in the group I intron core. J Mol Biol 2003, 329:229-238.
94. Takamoto K, Das R, He Q, Doniach S, Brenowitz M, Herschlag D,
Chance MR: Principles of RNA compaction: insights from the
equilibrium folding pathway of the P4–P6 RNA domain in
monovalent cations. J Mol Biol 2004, 343:1195-1206.
95. Takamoto K, He Q, Morris S, Chance MR, Brenowitz M:
Monovalent cations mediate formation of native tertiary
structure of the Tetrahymena thermophila ribozyme. Nat
Struct Biol 2002, 9:928-933.
96. Nesbitt S, Hegg LA, Fedor MJ: An unusual pH-independent and
metal-ion-independent mechanism for hairpin ribozyme
catalysis. Chem Biol 1997, 4:619-630.
97. Murray JB, Seyhan AA, Walter NG, Burke JM, Scott WG: The
hammerhead, hairpin and VS ribozymes are catalytically
proficient in monovalent cations alone. Chem Biol 1998,
5:587-595.
98. Travers KJ, Boyd N, Herschlag D: Low specificity of metal ion
binding in the metal ion core of a folded RNA. RNA 2007,
13:1205-1213.
99. Das R, Travers KJ, Bai Y, Herschlag D: Determining the Mg2+
stoichiometry for folding an RNA metal ion core. J Am Chem
Soc 2005, 127:8272-8273.
100. Bukhman YV, Draper DE: Affinities and selectivities of divalent
cation binding sites within an RNA tertiary structure. J Mol Biol
1997, 273:1020-1031.
101. Vieregg J, Cheng W, Bustamante C, Tinoco I Jr: Measurement of
the effect of monovalent cations on RNA hairpin stability. J Am
Chem Soc 2007, 129:14966-14973.
102. Tinoco I Jr, Li PT, Bustamante C: Determination of
thermodynamics and kinetics of RNA reactions by force. Q Rev
Biophys 2006, 39:325-360.
103. Li PT, Collin D, Smith SB, Bustamante C, Tinoco I Jr: Probing the
mechanical folding kinetics of TAR RNA by hopping, force-
jump, and force-ramp methods. Biophys J 2006, 90:250-260.
104. Li PT, Bustamante C, Tinoco I Jr: Real-time control of the
energy landscape by force directs the folding of RNA
molecules. Proc Natl Acad Sci U S A 2007, 104:7039-7044.
105 . Li PT, Bustamante C, Tinoco I Jr: Unusual mechanical stability
 of a minimal RNA kissing complex. Proc Natl Acad Sci U S A
2006, 103:15847-15852.
Using optical tweezers, the authors probe the energetics of the formation
and unfolding of a kissing-loop motif. They find that the stability of the
kissing-loop interaction is unusually strong.
106 . Greenleaf WJ, Frieda KL, Foster DA, Woodside MT, Block SM:
  Direct observation of hierarchical folding in single riboswitch
aptamers. Science 2008, 319:630-633.
Using optical tweezers, the authors follow the entire folding trajectory of a
simple adenine riboswitch aptamer. With extremely high accuracy, the
authors observe the formation of secondary and tertiary structure, as well
as the ligand-induced stabilization of the folded aptamer state, resolving
the hierarchical folding of this important regulatory ncRNA.
107. Woodside MT, Behnke-Parks WM, Larizadeh K, Travers K,
Herschlag D, Block SM: Nanomechanical measurements of the
sequence-dependent folding landscapes of single nucleic
acid hairpins. Proc Natl Acad Sci U S A 2006, 103:6190-6195.
108. Woodside MT, Anthony PC, Behnke-Parks WM, Larizadeh K,
Herschlag D, Block SM: Direct measurement of the full,
sequence-dependent folding landscape of a nucleic acid.
Science 2006, 314:1001-1004.
109 . Shcherbakova I, Mitra S, Beer RH, Brenowitz M: Fast Fenton
 footprinting: a laboratory-based method for the time-resolved
analysis of DNA, RNA and proteins. Nucleic Acids Res 2006,
34:e48.
The authors demonstrate the feasibility of rapid acquisition of structural
footprinting data at the millisecond time scale using hydroxyl radicals
generated using a chemical source and standard quench-flow mixers.
Such a technique makes hydroxyl radical footprinting techniques acces-
sible to the wide scientific community without the use of a synchrotron
source.
www.sciencedirect.com
Advances in RNA structure and function Herschlag and Chu 313
110. Das R, Laederach A, Pearlman SM, Herschlag D, Altman RB:
SAFA: semi-automated footprinting analysis software for
high-throughput quantification of nucleic acid footprinting
experiments. RNA 2005, 11:344-354.
111. Mitra S, Shcherbakova IV, Altman RB, Brenowitz M, Laederach A:
High-throughput single-nucleotide structural mapping by
capillary automated footprinting analysis. Nucleic Acids Res.
Advance Access published on May 13, 2008, DOI 10.1093/nar/
gkn267.
112. Wilkinson KA, Merino EJ, Weeks KM: Selective 20 -hydroxyl
acylation analyzed by primer extension (SHAPE): quantitative
RNA structure analysis at single nucleotide resolution. Nat
Protoc 2006, 1:1610-1616.
113. Wilkinson KA, Merino EJ, Weeks KM: RNA SHAPE chemistry
reveals nonhierarchical interactions dominate equilibrium
structural transitions in tRNA(Asp) transcripts. J Am Chem Soc
2005, 127:4659-4667.
114. Merino EJ, Wilkinson KA, Coughlan JL, Weeks KM: RNA structure
analysis at single nucleotide resolution by selective
20 -hydroxyl acylation and primer extension (SHAPE).
J Am Chem Soc 2005, 127:4223-4231.
115 . Laederach A, Shcherbakova I, Jonikas MA, Altman RB,
 Brenowitz M: Distinct contribution of electrostatics, initial
conformational ensemble, and macromolecular
stability in RNA folding. Proc Natl Acad Sci U S A 2007,
104:7045-7050.
The authors present a model for the folding landscape of a group I intron,
including folding intermediates and pathways, using the computational
analysis of a large body of footprinting data.
116. Laederach A, Shcherbakova I, Liang MP, Brenowitz M, Altman RB:
Local kinetic measures of macromolecular structure reveal
partitioning among multiple parallel pathways from the
earliest steps in the folding of a large RNA molecule. J Mol Biol
2006, 358:1179-1190.
117. Ha T, Zhuang X, Kim HD, Orr JW, Williamson JR, Chu S: Ligand-
induced conformational changes observed in single RNA
molecules. Proc Natl Acad Sci U S A 1999, 96:9077-9082.
118. Kim HD, Nienhaus GU, Ha T, Orr JW, Williamson JR, Chu S: Mg2+-
dependent conformational change of RNA studied by
fluorescence correlation and FRET on immobilized single
molecules. Proc Natl Acad Sci U S A 2002, 99:4284-4289.
119. Zhuang X, Bartley LE, Babcock HP, Russell R, Ha T, Herschlag D,
Chu S: A single-molecule study of RNA catalysis and folding.
Science 2000, 288:2048-2051.
120. Zhuang X: Single-molecule RNA science. Annu Rev Biophys
Biomol Struct 2005, 34:399-414.
121. Zhuang X, Kim H, Pereira MJ, Babcock HP, Walter NG, Chu S:
Correlating structural dynamics and function in single
ribozyme molecules. Science 2002, 296:1473-1476.
122 . Ye JD, Tereshko V, Frederiksen JK, Koide A, Fellouse FA,
 Sidhu SS, Koide S, Kossiakoff AA, Piccirilli JA: Synthetic
antibodies for specific recognition and crystallization
of structured RNA. Proc Natl Acad Sci U S A 2008,
105:82-87.
RNA structures are more difficult to obtain versus proteins. The authors
present a new method for RNA crystallization on the basis of the idea that
protein binding may help chaperone RNA crystal formation. These ideas
are demonstrated by the crystallization of the P4–P6 domain of Tetra-
hymena in complex with an antibody.
123. Das R, Kudaravalli M, Jonikas M, Laederach A, Fong R,
Schwans JP, Baker D, Piccirilli JA, Altman RB, Herschlag D:
Structural inference of native and partially folded RNA by high-
throughput contact mapping. Proc Natl Acad Sci U S A 2008,
105:4144-4149.
124. Das R, Baker D: Automated de novo prediction of native-like
RNA tertiary structures. Proc Natl Acad Sci U S A 2007,
104:14664-14669.
125. Getz M, Sun X, Casiano-Negroni A, Zhang Q, Al-Hashimi HM:
NMR studies of RNA dynamics and structural plasticity using
NMR residual dipolar couplings. Biopolymers 2007, 86:384-402.
Current Opinion in Structural Biology 2008, 18:305–314
314 Nucleic acids
126. Shajani Z, Varani G: NMR studies of dynamics in RNA and DNA
by 13C relaxation. Biopolymers 2007, 86:348-359.
127. Zhang Q, Sun X, Watt ED, Al-Hashimi HM: Resolving the
motional modes that code for RNA adaptation. Science 2006,
311:653-656.
128. Getz MM, Andrews AJ, Fierke CA, Al-Hashimi HM: Structural
plasticity and Mg2+ binding properties of RNase P P4 from
Current Opinion in Structural Biology 2008, 18:305–314
combined analysis of NMR residual dipolar couplings and
motionally decoupled spin relaxation. RNA 2007, 13:251-266.
129. Sun X, Zhang Q, Al-Hashimi HM: Resolving fast and slow
motions in the internal loop containing stem-loop 1 of
HIV-1 that are modulated by Mg2+ binding: role in the
kissing-duplex structural transition. Nucleic Acids Res 2007,
35:1698-1713.
www.sciencedirect.com